SOSS complexes participate in the maintenance of genomic stability

Abstract

Proteins that bind to single-stranded DNA (ssDNA) are essential for DNA replication, recombinational repair, and maintenance of genomic stability. Here, we describe the characterization of an ssDNA-binding heterotrimeric complex, SOSS (sensor of ssDNA) in human, which consists of human SSB homologs hSSB1/2 (SOSS-B1/2) and INTS3 (SOSS-A) and a previously uncharacterized protein C9orf80 (SOSS-C). We have shown that SOSS-A serves as a central adaptor required not only for SOSS complex assembly and stability, but also for facilitating the accumulation of SOSS complex to DNA ends. Moreover, SOSS-depleted cells display increased ionizing radiation sensitivity, defective G2/M checkpoint, and impaired homologous recombination repair. Thus, our study defines a pathway involving the sensing of ssDNA by SOSS complex and suggests that this SOSS complex is likely involved in the maintenance of genome stability.

Fig. 1. Formation of a SOSS complex containing INTS3 (SOSS-A), hSSB1/2 (SOSS-B1/2), and C9orf80 (SOSS-C)

(A) 293T cells stably expressing SFB-tagged (S-tag, Flag epitope tag, and streptavidin-binding peptide tag)-hSSB1 or hSSB2 were used for tandem affinity purification of protein complexes specifically from chromatin fractions isolated from irradiated cells. Tables are summaries of proteins identified by Mass spectrometry analysis. Letters in bold indicate the bait proteins. (B) HeLa cells were treated with ionizing radiation (10 Gy) or left untreated and then incubated for 2 hours before collection. Immunoprecipitations were performed using preimmune serum or anti-SOSS antibodies. Interactions among endogenous components of SOSS complex were assessed by immunoblotting using antibodies as indicated. (C) Heterotrimeric complex formation was studied by gel filtration chromatography as described in Materials and Methods. Aliquots from peak fractions were analyzed on 12.5% SDS-PAGE and confirmed by Western blot analysis. (D) Specific binding of SOSS-A to SOSS-B1 or SOSS-C. SF9 cells were co-infected with baculoviruses expressing GST or GST- tagged SOSS-A together with those expressing His-SOSS-B1 or SFB-SOSS-C. Immunoprecipitation and immunoblotting were carried out as indicated. (E) Schematic presentation of SOSS-A constructs used in this study. (F) Mapping of the corresponding regions required for SOSS-A/SOSS-B or SOSS-A/SOSS-C interaction. Immunoprecipitation reactions were performed using S protein beads and then subjected to Western blot analyses using antibodies as indicated. * Nonspecific band.

Fig. 2. SOSS-A regulates SOSS complex stability and focus formation

(A) Immunoblots showing levels of SOSS-A, SOSS-B1 and SOSS-B2 in lysates prepared from HeLa cells transfected with the indicated siRNAs. Two hours after mock treatment or ionizing radiation (10 Gy), cells were harvested and cell lysates were blotted with indicated antibodies. (B) Localization of SOSS components in response to IR in HeLa cells. Cells were irradiated and immunostained as described in Materials and Methods with anti-SOSS and pH2AX antibodies. (C, D). SOSS-A is required for the accumulation of SOSS-B1/2 at DNA damage sites. HeLa cells were transfected twice with either SOSS-A siRNA or a nontargeting control siRNA. 48 hours after the second transfection, cells were irradiated and immunostaining experiments were performed. Representative SOSS-B1/2 foci were shown (C). Quantification results were the average of three independent experiments and were presented as mean +/− SEM (D). More than one hundred cells were counted in each experiment. (E) SOSS-A is required for SOSS-B1/2 chromatin localization. HeLa cells depleted of endogenous SOSS-A were treated with 10 Gy IR or left untreated. Two hours later, cells were collected and chromatin fractions were isolated as described in Materials and Methods. Immunoblotting experiments were performed using indicated antibodies.

Fig. 3. SOSS-A is required for IR-induced G2/M checkpoint, cell survival and efficient DNA repair

(A) G2/M checkpoint defect was observed in SOSS-A-depleted cells. HeLa cells transfected with control or SOSS-A siRNAs were exposed to 0 or 2 Gy of IR. Cells were incubated for 1 hour before fixation and subjected to staining with antibody to phosphorylated histone H3 (pH3) and propidium iodide. The percentages of mitotic cells were determined by fluorescence-activated cell sorting analysis. The boxed area in the top left panel indicates mitotic cells. (B) Radiation sensitivity of cells lacking SOSS-A. HeLa cells were transfected with control or SOSS-A siRNAs and then irradiated with indicated doses of IR. Percentages of surviving colonies were determined two weeks later. These experiments were performed in triplicate, and the results represent the average of three independent experiments and were presented as mean +/− SEM. (C, D) SOSS-A promotes homologous recombination. U2OS cells with a single integration of DR-GFP were transfected twice with either SOSS-A siRNA or a nontargeting control siRNA. 24 hours after the second transfection, cells were electroporated with pCBASce construct and were subjected to FACS analyses 48 hours later (D). Results (mean +/− SEM) were the average of three independent experiments (C). (E, F) SOSS-A is required for efficient RAD51 foci formation. SOSS-A-depleted HeLa cells were treated with 10 Gy IR and recovery for 6 hours before fixation. Immunostaining experiments were performed as described in the in Materials and Methods. Representative RAD51 foci were shown (F). Quantification results were the average of three independent experiments and were presented as mean +/− SEM (E). More than one hundred cells were counted in each experiment.

Fig. 4. Both SOSS and CTIP/RPA promote optimal DNA damage response

(A) MRN complex is required for efficient SOSS complex foci formation. MRN-depleted HeLa cells were treated with 10 Gy IR and allowed to recover for 6 hours before fixation. Immunostaining experiments were performed as described in Materials and Methods. Representative SOSS foci were shown. (B) Foci formation of SOSS and RPA occurs independently. HeLa cells transfected with indicated siRNAs were treated with 10 Gy IR and allowed to recover for 6 hours before fixation. Immunostaining experiments were performed as described in Materials and Methods and representative SOSS and RPA2 foci were shown. (C) Simultaneous ablation of SOSS-A and CTIP resulted in further defect in HR. Experiments were carried out similar to that described in Figure 3C. Results (mean +/− SEM) were the average of three independent experiments. (D) Simultaneous depletion of SOSS-A and CTIP resulted in added IR sensitivity. Colony formation assays were performed as described in Materials and Methods and results (mean +/− SEM) are averages of three independent experiments. (E). A proposed model for the DNA-damage-responsive pathway involving SOSS complex.